Characterization of EPO N-Glycans using RapiFluor-MS and HILIC Profiling

Significance of the Topic

Recombinant human erythropoietin (rhEPO) is a cornerstone therapy for anemia management, but its function and half-life are heavily influenced by the structures of its N-linked glycans. Detailed profiling of these glycans is critical for biosimilar development, quality control, and ensuring consistent therapeutic performance.

Objectives and Study Overview

This study aimed to develop a streamlined workflow for rapid release, labeling, and characterization of EPO N-glycans using Waters GlycoWorks RapiFluor-MS chemistry coupled with hydrophilic interaction chromatography (HILIC) and high-resolution mass spectrometry. The goal was to achieve comprehensive glycan profiling in under 30 minutes of sample preparation with high sensitivity and resolution.

Methodology and Instrumentation

The approach leveraged a fast enzymatic deglycosylation protocol accelerated by RapiGest SF surfactant and Rapid PNGase F at 50 °C. Released glycosylamines were then tagged in five minutes with RapiFluor-MS, enhancing fluorescence yield and electrospray ionization efficiency. Labeled glycans were purified via HILIC µElution plates and eluted for direct analysis.

Použitá instrumentace:

• GlycoWorks RapiFluor-MS N-Glycan Kit
• RapiGest SF surfactant and Rapid PNGase F enzyme
• HILIC µElution plate and SPE Elution Buffer
• ACQUITY UPLC I-Class System with Glycan BEH Amide column (2.1×150 mm, 1.7 µm, 130 Å)
• Xevo G2-S QTof mass spectrometer with positive mode ESI detection

Key Results and Discussion

HILIC-FLR-MS chromatograms revealed well-resolved peaks corresponding to various tetra-antennary N-glycans bearing three to four sialic acids and poly-N-acetyl lactosamine extensions. The predominant species, FA4G4S4, accounted for ~20 % of the glycan pool. Minor species included mono- and di-lactosamine-extended glycans (FA4G4Lac1S4 at ~12 % and FA4G4Lac2S4 at ~4.5 %), as well as tri- and tetra-lactosamine variants at trace levels (0.75 % and 0.25 %, respectively). High mass accuracy (<5 ppm) and enhanced sensitivity enabled confident assignments without multidimensional fractionation.

Benefits and Practical Applications

The RapiFluor-MS workflow compresses glycan sample preparation to 30 minutes while boosting detection limits through improved fluorescence and MS response. The single-dimension HILIC separation simplifies analysis and accelerates throughput, offering an efficient platform for biosimilar comparability studies, lot release testing, and detailed quality attribute monitoring in biopharmaceutical manufacturing.

Future Trends and Possibilities

Advancements may include integration with ultra-high-throughput automation, coupling with ion mobility for enhanced glycan isomer separation, and expansion to O-glycan analysis. Machine-learning-driven spectral interpretation could further streamline data processing and accelerate regulatory submissions for next-generation glycoprotein therapeutics.

Conclusion

The combination of rapid RapiFluor-MS labeling and HILIC-ESI-QTof-MS offers a sensitive, high-resolution method for characterizing complex EPO N-glycans. This single-dimension approach supports fast, reliable glycan profiling essential for therapeutic development and quality assurance of erythropoietin products.

Reference

1. Dube S, Fisher JW, Powell JS. Glycosylation at specific sites of erythropoietin is essential for biosynthesis, secretion, and biological function. J Biol Chem. 1988;263(33):17516–21.
2. Sasaki H, Bothner B, Dell A, Fukuda M. Carbohydrate structure of erythropoietin expressed in Chinese hamster ovary cells by a human erythropoietin cDNA. J Biol Chem. 1987;262(25):12059–76.
3. Hashii N, Harazono A, Kuribayashi R, et al. Characterization of N-glycan heterogeneities of erythropoietin products by LC/MS and multivariate analysis. Rapid Commun Mass Spectrom. 2014;28(8):921–32.
4. Takeuchi M, Inoue N, Strickland TW, et al. Relationship between sugar chain structure and biological activity of recombinant human erythropoietin. Proc Natl Acad Sci USA. 1989;86(20):7819–22.
5. Bones J, McLoughlin N, Hilliard M, et al. 2D-LC analysis of BRP 3 erythropoietin N-glycosylation using anion exchange and HILIC-UPLC reveals long poly-N-acetyl lactosamine extensions. Anal Chem. 2011;83(11):4154–62.
6. Lauber MA, Yu YQ, Brousmiche DW, et al. Rapid preparation of released N-glycans for HILIC analysis using a labeling reagent that facilitates sensitive fluorescence and ESI-MS detection. Anal Chem. 2015;87(10):5401–8.